Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP‐dependent manner

Ribosomes complexed with synthetic mRNA and peptidyl‐tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis. The peptide‐releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non‐hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide. With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide. RF3 hydrolysis of GTP to GDP + Pi was dependent only on ribosomes and not on RF1 or RF2. RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release. A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.

[1]  M. Ehrenberg,et al.  Rate of translation of natural mRNAs in an optimized in vitro system. , 1996, Archives of biochemistry and biophysics.

[2]  C. Caskey,et al.  Peptide chain termination: effect of protein S on ribosomal binding of release factors. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Ganoza Mc.  Polypeptide chain termination in cell-free extracts of E. coli. , 1966 .

[4]  M. Capecchi,et al.  Characterization of three proteins involved in polypeptide chain termination. , 1969, Cold Spring Harbor symposia on quantitative biology.

[5]  I. Stansfield,et al.  Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast , 1996, Molecular microbiology.

[6]  S Thirup,et al.  Crystal Structure of the Ternary Complex of Phe-tRNAPhe, EF-Tu, and a GTP Analog , 1995, Science.

[7]  P. Jelenc,et al.  Rapid purification of highly active ribosomes from Escherichia coli. , 1980, Analytical biochemistry.

[8]  Y. Nakamura,et al.  Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Soffientini,et al.  Purification procedure for bacterial translational initiation factors IF2 and IF3. , 1994, Protein expression and purification.

[10]  P. Siekevitz,et al.  Ribosomes and protein synthesis , 1981, The Journal of cell biology.

[11]  R. Buckingham,et al.  Osmo-expression and fast two-step purification of Escherichia coli translation termination factor RF-3. , 1995, European journal of biochemistry.

[12]  L. Isaksson,et al.  Emerging Understanding of Translation Termination , 1996, Cell.

[13]  M. Grunberg‐Manago,et al.  Translational regulation of the Escherichia coli threonyl-tRNA synthetase gene: structural and functional importance of the thrS operator domains. , 1993, Biochimie.

[14]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[15]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[16]  A. Liljas,et al.  Three‐dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. , 1994, The EMBO journal.

[17]  S M Block,et al.  Fifty Ways to Love Your Lever: Myosin Motors , 1996, Cell.

[18]  G. Spedding.  Ribosomes and protein synthesis : a practical approach , 1990 .

[19]  U. Maitra,et al.  Protein factors involved in polypeptide chain initiation in Escherichia coli. , 1969, Cold Spring Harbor symposia on quantitative biology.

[20]  Y. Kaziro.  The role of guanosine 5'-triphosphate in polypeptide chain elongation. , 1978, Biochimica et biophysica acta.

[21]  L. Mora,et al.  Localization and characterization of the gene encoding release factor RF3 in Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[22]  C. Kurland,et al.  Nucleoside triphosphate regeneration decreases the frequency of translation errors. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. Ehrenberg,et al.  Mutations in 23 S ribosomal RNA perturb transfer RNA selection and can lead to streptomycin dependence. , 1994, Journal of molecular biology.

[24]  M. Rodnina,et al.  Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome , 1997, Nature.

[25]  M. Capecchi.  Polypeptide chain termination in vitro: isolation of a release factor. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[26]  E. Scolnick,et al.  Peptide chain termination. 3. Stimulation of in vitro termination. , 1969, Proceedings of the National Academy of Sciences of the United States of America.

[27]  L. Kisselev,et al.  Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. , 1995, The EMBO journal.

[28]  R. Buckingham,et al.  Function of Polypeptide Chain Release Factor RF-3 in Escherichia coli , 1995, The Journal of Biological Chemistry.

[29]  O. Jean-Jean,et al.  Overexpression of human release factor 1 alone has an antisuppressor effect in human cells , 1997, Molecular and cellular biology.

[30]  E. Scolnick,et al.  Peptide chain termination, codon, protein factor, and ribosomal requirements. , 1969, Cold Spring Harbor symposia on quantitative biology.

[31]  K. Mullis,et al.  Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. , 1986, Cold Spring Harbor symposia on quantitative biology.

[32]  J. Wang,et al.  The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution. , 1994, The EMBO journal.

[33]  L. Kisselev,et al.  Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. , 1996, RNA.

[34]  K. Holmes,et al.  Muscle proteins--their actions and interactions. , 1996, Current opinion in structural biology.

[35]  T. Seno,et al.  Purification of Escherichia coli methionine tRNAF and methionine tRNAM and studies on their biophysical and biochemical properties , 1968 .

[36]  C. Kurland,et al.  Release factor-dependent false stops are infrequent in Escherichia coli. , 1993, Journal of molecular biology.

[37]  C. Gualerzi,et al.  Selection of the mRNA translation initiation region by Escherichia coli ribosomes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M. Nirenberg,et al.  Release factors differing in specificity for terminator codons. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Y. Nakamura,et al.  Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[40]  M. Nirenberg,et al.  Sequential Translation of Trinucleotide Codons for the Initiation and Termination of Protein Synthesis , 1968, Science.